CN103477994A - Bacterial strain used for producing ganoderma lucidum polysaccharides by complete feed liquid fermentation of rice bran and wheat bran - Google Patents
Bacterial strain used for producing ganoderma lucidum polysaccharides by complete feed liquid fermentation of rice bran and wheat bran Download PDFInfo
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Abstract
The invention belongs to the field of microbial application technology and food biotechnology, and discloses a ganoderma lucidum strain used for producing polysaccharides by complete feed liquid fermentation of rice bran and wheat bran. Ganoderma lucidum JSU6161314 is stored in China Center for Type Culture Collection (CCTCC) on 25th, June, 2013, preservation number is CCTCC M2013287, and the bacterial strain is named as Ganoderma lucidum CCTCC M2013287. According to a method disclosed in a drawing of the invention, the novel bacterial strain, which is capable of realizing high yield of ganoderma lucidum polysaccharides, is obtained by modifying ganoderma lucidum CFCC6043. The novel bacterial strain is used for rapid fermentation in a rice bran and wheat bran complete feed liquid culture medium and high yield of ganoderma lucidum polysaccharides. After fermentation, hypha dry weight and hypha polysaccharide yield increase 23.0% and 45% respectively compared with that of the original strain.
Description
Technical field
The present invention relates to the food microorganisms applied technical field, relate in particular to a strain and be adapted in rice bran and the composite liquefied medium of wheat bran complete feed the also new mutagenic strain of the ganoderma strain capable of high yield GL-B of growing.
Background technology
Since the people such as Chihara (list of references: Chihara G, MaedaY, Hamuro J, et al.Inhibition of mouse sarcoma180by polysaccharides from Lentinus edodes (Berk.) sing[J] .Nature, 1969,222 (5194): 687-688.) in 1969, on " Nature ", publish thesis, since the announcement lentinan has antitumor activity, have been found that the medicinal fungi with antitumor activity reaches kind more than 200, wherein a part or edible fungus, as grifola frondosus, Hericium erinaceus, glossy ganoderma etc.Can produce the composition such as the various active such as amino acid, protein, vitamin, polysaccharide, glycoside, flavonoids and antibiotic or Rich in Trace Element selenium, zinc etc. in the mycelium of these edible mushrooms, fruit body, sclerotium or spore, have improve body immunity, antitumor, strengthen liver function, the multiple efficacies such as anti-oxidant.The existing edible mushroom health-care food in domestic market as ganoderma lucidum capsule, grifola frondosus capsule and capsule of Chinese caterpillar fungus etc. in fast sale.New Zealand, Japan, the U.S. etc. all produce similar product are arranged in the world.Zelanian " GanoPoly " series of products are formed by the polysaccharide composite shitosan of the extractions such as glossy ganoderma, rainbow conk, are mainly used in improving the aspects such as immunity, auxiliary antineoplaston.With regard to GL-B, research for it shows: GL-B has the curative effects such as good raising immunity, auxiliary antineoplaston, the test of pesticide effectiveness of extracting GL-B such as Huang, Seto etc. from the glossy ganoderma thalline shows, GL-B has immunoregulation effect and antitumor action, and (list of references is shown in 1. Huang S.Q., Ning Z.X.Extraction of polysaccharide from Ganoderma lucidum and its immune enhancement activity[J] .Int J Biol Macromol, 2010,47 (3): 336～341; 2. Seto S.W., Lam T.Y, Tam H.L., et al.Novel hypoglycemic effects of Ganoderma lueidum water-extract in obese/diabetic (db/+db) mice[J] .Phytomedicine, 2009, (16): 426～436; 3. Hikino H, Konno C, Mirin Y, et al.Isolation and hypoglycemic activity of ganoderans A and B, glycans of Ganoderma lucidum fruit bodies[J].Planta?Med，1985，(12)：339～340)
Glossy ganoderma (Ganoderma luccidum) another name sesame grass, auspicious grass, it is Eumycota, Basidiomycotina, Hymenomycetes, Aphyllophorales, the glossy ganoderma Cordycepps, the Ganoderma fungi, mainly be distributed in the torrid zone and the subtropical zone in Asia, Australia, Africa and America, and minority also is distributed in Temperate Region in China, only there are 4 kinds of Ganoderma in the Europe that is located in temperate zone, the Northern Hemisphere, and about 5 kinds of North America.China extends across the torrid zone to cool temperate zone, and the Ganodermataceae kind is many and distribute extensively, is one of country that world's Ganoderma fungal species diversity is the abundantest.Traditionally, the obtain manner of glossy ganoderma is mainly artificial cultivation or field acquisition, with fruit body or conidial powder, is used as medicine.But the Ganoderma Lucidum cycle is long, production efficiency is low, labour intensity is large, is subject to again the restrictions such as season, environment, subjects to damage by disease and insect, and quality and output are unstable, and field acquisition glossy ganoderma limited amount is difficult to meet large production requirement.Edible fungus liquid submerged fermentation technology, have obvious advantage than traditional fruit body cultivation.For the High-efficient Production GL-B, the researcher attaches great importance to the research of the liquid cultivating method to promoting suitability for industrialized production, has obtained many achievements in research.Hoe Xiaoyan, Zeng Xi and other liquid culture of Ganoderma conditions were studied, the main contents of such research involves the production strain, medium composition and fermentation parameters, will reduce production costs and increase yields of polysaccharide as a research priority.Wherein most process using as starch, potato, glucose etc. as medium component, even if use the agro-product processing by product as rice bran or wheat bran, be also using it as submember, be still that to take grain class raw material, glucose or other raw materials be main Carbon and nitrogen sources.
China is large agricultural country, and the agricultural byproducts source is abundant.Rice bran, wheat bran are as the accessory substance of paddy and wheat processing, and not only its nutrient component is abundant, and cheap.Be rich in the materials such as starch, cellulose in rice bran, and wheat bran is rich in the materials such as albumen, cellulose.Theoretically, rice bran and wheat bran are compound has possessed the glossy ganoderma needed Carbon and nitrogen sources material of growing.Under the effect of glossy ganoderma self cellulase and other enzymes, glossy ganoderma can change into rice bran and wheat bran the nutriment of self and be grown, and produces GL-B.Therefore, use the cheap agricultural byproducts such as rice bran, wheat bran, the required nutriments of Ganoderma lucidum submerged fermentation such as complete place of glucose, produce the ganodermal drop with auxiliary oncotherapy, high-valued application to the low side raw material is significant, has high economic worth.
According to inventor herein Liu Wei people seminar years of researches experience, some edible mushroom strainses upgrowth situation on the rice bran that does not add glucose or other grain class raw materials or wheat bran liquid culture medium is very desirable, need to do specially treated as induction mutation of bacterium to obtain good growth.For example, Liu Weimin instructs several Master degree candidates to carry out deep research to edible mushroom grifola frondosus liquid state fermentation rice bran or wheat bran, having obtained some achievements, formed as Publication about Document: (1) Yang Suohua. the grifola frondosus ferment rice bran prepares polysaccharide [D]. master thesis. Zhenjiang: the .2006 of Jiangsu University; (2) Gu Huimin. grifola frondosus is liquid research [D] of cultivating product polysaccharide and enrichment Organic Selenium in the rice bran medium. master thesis. Zhenjiang: the .2009 of Jiangsu University; (3) to open and build. Physical mutagenesis grifola frondosus liquid fermentation rice bran wheat bran produces the research [D] of polysaccharide. master thesis. Zhenjiang: Jiangsu University, 2010; (4) Guo Chunmei. the induction mutation of bacterium of grifola frondosus, liquid fermentation rice bran wheat bran produce polysaccharide and rich selenium research [D]. master thesis. Zhenjiang: Jiangsu University, 2011; (5) Li Yongzhuan. transform rice bran and the Grifola frondosa strain mutagenesis of wheat bran high polysaccharide and the fermentation test [D] of existing bacterium. master thesis. Zhenjiang: Jiangsu University, 2012; (6) Liu Weimin, open and build, Guo Chunmei, etc. for the Grifola frondosa strain [P] of rice bran and wheat bran compound material production polysaccharide, 10579078.5,2010; (7) Liu Weimin, open and build, Guo Chunmei, etc. use rice bran wheat bran compound material and grifola frondosus mutagenic strain to produce the method [P] of polysaccharide, 1010579048.4,2010; (7) Liu Weimin, Guo Chunmei, open and build, etc. for the bacterial strain [P] of ferment rice bran and wheat bran extract production grifolan, 10150888.3,2011 (applications); (8) Li Yanan. the mutagenesis of grifola frondosus bacterial classification and fermentation and product property research [D]. master thesis. Zhenjiang: Jiangsu University, 2013 (this paper publication date is in June, 2013, with the application of this patent, submits to the days identical); (9) Zhao Li. the research [D] of grifola frondosus liquid state fermentation enrichment manganese in rice bran wheat bran complex medium. master thesis. Zhenjiang: Jiangsu University, 2012; (10) Liu Lili. high value transforms Ganoderma lucidum submerged fermentation and the induction mutation of bacterium [D] of rice bran wheat bran. master thesis.Zhenjiang: Jiangsu University, 2012; (11) Guo Tianlong. the full rice bran wheat bran of the high-valued conversion of lucidum strain mutagenesis and liquid state fermentation research [D]. master thesis. Zhenjiang: Jiangsu University, 2013 (this paper publication date is in June, 2013, with the application of this patent, submits to the days identical).
From above-mentioned list of references, the inventor herein Liu Wei people are studied around efficient high-valued this special topic of rare edible fungi polysaccharide that is converted into of rice bran wheat bran always, realize a plurality of innovation and creation imaginations, and proposed to carry out rice bran wheat bran complete feed liquid fermentation with glossy ganoderma, the Research Thinking that efficient high-valued conversion rice bran wheat bran is GL-B, to original strain mutagenic treatment in addition, instructing the Liu Lili mutagenesis testing unsuccessful, (be negative mutagenesis, see the analysis of Liu Lili Master's thesis p46 his-and-hers watches 5.2, the Master's thesis of Liu Lili all adopts the rice bran wheat bran to cross leaching juice and makes medium, rice bran wheat bran complete feed liquid culture medium of the present invention) basis on, summing up experience, instruct again Guo Tianlong to carry out systematic research, obtained positive mutagenic strain, and mutagenic strain liquid state fermentation rice bran wheat bran complete feed newly proposed, the method that efficient high-valued conversion rice bran wheat bran is GL-B, the content of another patent of invention that has formed patent of the present invention and declared simultaneously.Master's thesis publication date of Guo Tianlong is in June, 2013, with the application of this patent, submits to days identical, does not affect novelty of the present invention.
One of key issue of the present invention is for must obtain the new bacterial strain of a kind of glossy ganoderma, and this new bacterial strain can effectively be grown on rice bran wheat bran complete feed liquid culture medium and Efficient Conversion rice bran wheat bran complete feed is GL-B.The foundation of new bacterial strain and new bacterial strain all need innovation to the mode of newly utilizing of rice bran wheat bran, being suitable for the rice bran of new ganoderma strain capable and the proportioning of wheat bran composition liquid nutrient medium and the process conditions of liquid state fermentation all needs recent studies on to draw, have not been reported, therefore the present invention has uniqueness, innovation and practicality, possesses the essential characteristic of patent of invention.
The physics selection of microorganism mainly contains ultraviolet mutagenesis, microwave irradiation, ion beam mutagenesis etc. at present.For mutagenesis screening goes out to be adapted on full rice bran wheat bran complex medium to grow and produces the ganoderma strain capable of polysaccharide, the present invention adopts simple new approaches of physical mutagenesis, carry out ultraviolet mutagenesis and microwave irradiation test simultaneously, enlarge the site scope of tested glossy ganoderma starting strain sudden change, improve the possibility that obtains the direct mutation bacterial strain.Mutagenesis of the present invention obtains is adapted on full rice bran wheat bran complex medium growing and the bacterial strain that produces GL-B obtains for invention first.
Summary of the invention
The present invention to use soil and reduces the production cycle in order to reduce, reduce production costs, by low side raw material high-valued trans-utilization in addition, finally can reduce the GL-B product price, benefit masses, the by product rice bran of the consideration large agricultural product paddy of employing and wheat processing and wheat bran, as medium, no longer add other carbon sources as glucose and nitrogenous source, carry out the liquid fermentation of glossy ganoderma, produce GL-B.Realize this purpose, need mutagenesis screening to go out the ganoderma strain capable of suitable growth on rice bran wheat bran complete feed liquid culture medium.Therefore, the present invention will be take high polysaccharide as index by the method for ultraviolet, microwave irradiation, and the directed rice bran wheat bran that transforms be basic, select high yield, ganoderma strain capable cheaply.By the foundation of the screening techniques such as a series of stability, inheritance, guarantee the merit of glossy ganoderma, for further utilizing on a large scale the GL-B of rice bran and wheat bran production high value cheaply, lay the foundation.
The technical solution used in the present invention is as follows:
The present invention is set out by the glossy ganoderma CFCC6043 (Ganoderma lucidum CFCC6043) of China Forest microorganism fungus kind preservation administrative center (CFCC), mutagenesis screening by ultraviolet and microwave, a kind of new glossy ganoderma mutagenic strain glossy ganoderma JSU6161314 (Ganoderma lucidum JSU6161314) is provided, can there is high growth rates and high polysaccharide yield not adding on the rice bran wheat bran complete feed liquid culture medium of other Carbon and nitrogen sources; This glossy ganoderma JSU6161314 has been deposited in Wuhan, China Wuhan University Chinese Typical Representative culture collection center (CCTCC) on June 25th, 2013, preservation strain is numbered CCTCC M2013287, and name is called glossy ganoderma CCTCC M2013287 (Latin is called Ganoderma lucidum CCTCC M2013287).
In one aspect of the invention, provide the purposes of above-mentioned glossy ganoderma CCTCC M2013287, for ferment rice bran wheat bran complete feed liquid culture medium, produce GL-B.
Beneficial effect of the present invention
The present invention adopts simple physical mutagenesis technology ultraviolet and microwave irradiation technology seed selection glossy ganoderma superior strain, by laboratory, existing glossy ganoderma CFCC6043 sets out, carry out the parallel mutagenesis of ultraviolet and microwave, with growth rate, mycelia dry weight and exocellular polysaccharide are that index is screened, the final glossy ganoderma CCTCC M2013287 that obtains, produce glossy ganoderma and produce polysaccharide in not adding the rice bran wheat bran complete feed liquid culture medium of other Carbon and nitrogen sources, with original strain glossy ganoderma CFCC6043, to compare output higher, while being fermented by tank on microwave irradiation bacterial strain obtained strains glossy ganoderma CCTCC M2013287, mycelia dry weight and mycelia polysaccharide productive rate output are 6.3g/100mL medium and 0.203mg/100mL medium, starting strain glossy ganoderma CFCC6043 mycelia dry weight and mycelia polysaccharide output are 5.12g/100mL medium and 0.14mg/100mL medium, the mycelia dry weight of mutagenic strain glossy ganoderma CCTCC M2013287 fermentation and mycelia dry weight and the mycelia polysaccharide output of mycelia polysaccharide productivity ratio starting strain glossy ganoderma CFCC6043 have increased respectively 23.0% and 45%.Bacterial strain and starting strain after the antagonistic effect proof mutagenesis of glossy ganoderma CCTCC M2013287 and starting strain glossy ganoderma CFCC6043 are different on hereditary capacity, during screening, mutagenic strain glossy ganoderma CCTCC M2013287 growth characteristics are obviously faster than starting strain glossy ganoderma CFCC6043, thereby can judge that mutagenic strain is as desirable new bacterial strain.This new bacterial strain glossy ganoderma CCTCC M2013287 obtains for pioneering, belongs to a kind of new microbial resources, has uniqueness, novelty.Take new bacterial strain glossy ganoderma CCTCC M2013287 as basis, formed the method for new ferment rice bran wheat bran complete feed liquid culture medium, the method has changed in the past the rice bran wheat bran and has crossed after leaching juice or cellulase are processed and get juice and make the low shortcoming of rice bran wheat bran availability, efficiently utilized cheap rice bran wheat bran, can reduce production costs, reduce resource consumption, increase output.Described mutagenic fungi glossy ganoderma CCTCC M2013287 is under identical rice bran wheat bran usage amount condition, the mycelia dry weight of mutagenic strain glossy ganoderma CCTCC M2013287 fermentation and mycelia dry weight and the mycelia polysaccharide output of mycelia polysaccharide productivity ratio starting strain glossy ganoderma CFCC6043 have increased respectively 23.0% and 45%, obvious effect of increasing production.The present invention has produced the beneficial effect of technological progress.
Final products are the GL-B with effects such as strengthening immunity, auxiliary oncotherapy, can become the useful product of popular health care.By the high-valued trans-utilization of low side raw material, cost and resource have been saved.Therefore, the present invention has good social benefit.
The end product GL-B at present on market price higher, be high-valued product, the present invention has good economic worth.
To sum up, the present invention has possessed uniqueness, creativeness and the practicality of patent of invention, has produced useful technology, society and economic effect.
The accompanying drawing explanation
The flow chart that Fig. 1 is the parallel mutagenic breeding method of bacterial strain ultraviolet microwave of the present invention;
Fig. 2 is the ultraviolet irradiation effect, wherein annotates: the left side is for irradiating 0s, and the right is for irradiating 90s;
Fig. 3 is the microwave irradiation effect, wherein annotates: the left side is for irradiating 0s, and the right is for irradiating 10s;
Fig. 4 is the strain growth state, wherein annotates: be followed successively by from left to right ultraviolet No. 9, glossy ganoderma CCTCC M2013287, starting strain;
Fig. 5 is that the strain liquid seed morphology compares, and wherein annotates: be followed successively by from left to right starting strain, No. 9, ultraviolet, glossy ganoderma CCTCC M2013287;
The antagonism figure that Fig. 6 is mutagenic fungi glossy ganoderma CCTCC M2013287 and starting strain glossy ganoderma CFCC6043, wherein annotate: the left side is original strain glossy ganoderma CFCC6043, and the right is mutagenic fungi glossy ganoderma CCTCC M2013287.
Embodiment
The present invention is the flow process shown in accompanying drawing 1 to specifications, provide ultraviolet and microwave irradiation seed selection adding the method that has the ganoderma strain capable of high growth rates and high polysaccharide yield on the rice bran wheat bran complex medium of other Carbon and nitrogen sources, described method comprises the following steps:
Getting the existing glossy ganoderma in laboratory is starting strain;
Ganoderma strain capable is inoculated on the solid slant medium and normally cultivates;
Thalline is cultivated rear physiological saline wash-out and is made spore suspension;
Spore suspension obtained above is irradiated under uviol lamp and after microwave carries out mutagenesis, unglazed dark cultivation, primary screening goes out very fast, the more stable bacterial strain of growth rate;
On full rice bran, wheat bran liquid fermentation medium, fermented, the bacterial strain of postsearch screening high growth rates and high polysaccharide yield;
Carry out stability and inheritance analysis and identification;
In one embodiment, solid slant medium used is potato 200g/L, glucose 20g/L, peptone 5g/L, potassium dihydrogen phosphate 1.5g/L, magnesium sulfate 0.75g/L, Cobastab
110mg/L, agar 20g/L, pH nature.
In one embodiment, described constant temperature is 28 ℃.
In one embodiment, described ultraviolet mutagenesis adopts ruddiness secretly to operate, and the ultraviolet lamp tube that two power of usining are 30W, as the light source of ultraviolet mutagenesis, apart from 20cm, irradiates 30s.
In one embodiment, described unglazed dark cultivation is unglazed cultivation 2-3 days under 28 ℃.
In one embodiment, described unglazed dark cultivation used medium is rice bran, wheat bran solid plate medium: rice bran 20g/L, wheat bran 30g/L, potassium dihydrogen phosphate 1.5g/L, magnesium sulfate 0.75g/L, Cobastab
110mg/L, agar 20g/L, pH nature.
In one embodiment, described screening technique is the plate diameter determination method.
In one embodiment, described microwave irradiation adopts ruddiness secretly to operate, microwave power 700W, pulse frequency 2450Hz, mutation time 20s.
In one embodiment, described primary screening step is: from ultraviolet mutagenesis, microwave irradiation is unglazed dark culture plate, the well-grown single bacterium colony of picking is seeded to respectively in new rice bran wheat bran solid plate medium, pick out 10 strain fast growths and dense mutagenic fungi, to its cultivation of going down to posterity, therefrom select that 3 strains are fast with respect to starting strain growth, shapeliness, bacterial strain that stability is high.
In one embodiment, described secondary fermentation screening step is: the definite high growth rates variant of first secondary growth, as the object of postsearch screening, screens the bacterial strain of definitive variation strain merit stably express thereby ferment together with starting strain.First bacterium and starting strain are carried out to the shake flask fermentation test, continuously fermented for 5 generations, by index, determine the purpose mutagenic fungi.
In one embodiment, described shaking flask is the 250mL conical flask.
In one embodiment, described rice bran, wheat bran liquid fermentation medium are: rice bran 20g/L, wheat bran 30g/L (it is complete utilization that rice bran, wheat bran poach 4h do not get juice), potassium dihydrogen phosphate 1.5g/L, magnesium sulfate 0.75g/L, Cobastab
110mg/L, the pH nature.
In one embodiment, described index is exocellular polysaccharide, mycelia (mixing a small amount of raw material) dry weight and mycelia polysaccharide.
Transform the Analysis and Identification method of the glossy ganoderma mutant strain of rice bran and wheat bran compound material in the present invention, described method comprises the following steps:
The colonial morphology comparison;
Stability analysis;
In one embodiment, described is the colonial morphology comparison, to on inclined-plane, set out ganoderma strain capable and JSU61613 inoculation to the solid slant medium, each 6 repetitions, in 28 ℃ of constant incubators, cultivate 7 days, observe every day once, observe the growth rate that comprises bacterium colony, color, colony shape, smell etc.
In one embodiment, described stability analysis is for take mycelial growth rate as index investigation stability, finishing screen is selected to preferably dissociant of 10 strains, through 5 times, go down to posterity, select optimum JSU6161314 bacterial strain, this strain dissociant was gone down to posterity for 3 generations again, observe the variation of mycelial growth rate, thereby determine stability, and contrast with starting strain.
In one embodiment, described inheritance analysis is the antagonism analysis.
Embodiment 1 glossy ganoderma ultraviolet ray-microwave irradiation
1. the preparation of spore suspension
Glossy ganoderma is seeded in to 9cm and has gone out on the solid culture medium flat board of bacterium after cultivating 4d and rinse by the 10mL stroke-physiological saline solution, with the writing brush of sterilizing brush trophosome back and forth, collect washing lotion and get final product.
2. the selection of ultraviolet mutagenesis effect curve and ultraviolet mutagenesis dosage
Get 20 9cm plates, every ware is equipped with respectively freshly prepd spore suspension 5mL.Every two is one group, and being divided into is 10 groups.Irradiate respectively 0,5,10,15,20,25,30,45,60,150s (the 90s radiation response is shown in accompanying drawing 2), then every ware is got 1mL and suitably is coated with after dilution dull and stereotyped.Dull and stereotyped 28 ℃ of insulating boxs cultivations that are placed on dark black out with the black cloth parcel.After flat board grows bacterium colony, colony counting.Every group of mean value of usining two ware clump counts is as the clump count of this group.Calculate the mutagenesis lethality rate, take the mutagenesis lethality rate as index selection ultraviolet mutagenesis dosage.
In formula, A is bacterium colony regeneration number after mutagenesis; B is clump count before mutagenesis.
3. ultraviolet mutagenesis
Open the about 20min of ultraviolet preheating, cut-off footpath 9cm sterile petri dish 2 covers, add respectively the above-mentioned spore suspension 5mL adjusted, and put on electromagnetic shaker, the open plate lid, be 20cm in distance, under the uviol lamp that power is 30W, by the best exposure dose of above-mentioned selection, irradiates respectively.Cover the ware lid, close uviol lamp.During dose meter, from uncapping, only add a cover.First drive the electromagnetic shaker switch, then the irradiation of uncapping, make cell in spore suspension accept to irradiate even etc.
4. microwave irradiation
The spore suspension that absorption makes, inject the smooth plate in bottom, and the suspension amount of each plate is 10mL, the adjustment microwave power is 700W, pulse frequency is 2450Hz, according to the different processing times, spore suspension is carried out to radiation treatment (the 10s radiation response is shown in accompanying drawing 3).Draw spore suspension 0.3mL, coating rice bran, wheat bran solid plate medium, then be placed in 28 ℃ of insulating boxs and cultivate 3d.Count plate, calculate lethality rate.10 batches of continually mutagenizes, select on the rice bran bran mass can grow first out, healthy and strong, pure bacterial strain.
4. the screening of mutagenic strain
Get the bacteria suspension 0.2mL of ultraviolet irradiation, microwave, be coated with rod with aseptic glass and fill equably rice bran, wheat bran solid plate media surface, every batch is coated with 5 plates, 10 batches of mutagenesis, single bacterium colony of regeneration is seeded to respectively in new plate, pick out 10 strain fast growths and dense mutagenic fungi, to its cultivation of going down to posterity, therefrom select that 3 strains are fast with respect to starting strain growth, shapeliness, bacterial strain that stability is high.
Above-mentioned glossy ganoderma mutagenic fungi is carried out to the shake flask fermentation test, and take exocellular polysaccharide, mycelia, to mix dry weight be index, filters out the purpose mutagenic fungi.
Table 1 is selected bacterial strain, and each mixes dry weight for fermented hypha
Table 2 is selected each generation fermentation exocellular polysaccharide of bacterial strain
Can find out that from table 1 and 2 mycelia of dissociant JSU-10 (being glossy ganoderma JSU6161314) first generation fermentation and polysaccharide yield are all higher than starting strain, but in the second generation and the third generation, certain variation has occurred in its proterties, and glossy ganoderma JSU6161314 is relatively stable, its mycelia mixing dry weight and exocellular polysaccharide are all higher than starting strain, its third generation mycelia mixes dry weight and yield of extracellular polysaccharide has improved respectively 1.8% and 5.9% in the 100mL shaking flask is cultivated, glossy ganoderma JSU6161314 is deposited in Chinese Typical Representative culture collection center (CCTCC) on June 25th, 2013, preservation strain is numbered CCTCC M2013287, name is called glossy ganoderma CCTCC M2013287 (Latin is called Ganoderma lucidum CCTCC M2013287).
Test the analysis of a dissociant
1, biomorph Epidemiological Analysis
Lucidum strain on inclined-plane is inoculated on the solid slant medium, 6 repetitions, in 28 ℃ of constant incubators, cultivate 7 days, observe once every day, and the results such as the growth rate of observable bacterium colony, color, colony shape, smell are: glossy ganoderma CCTCC M2013287 growth rate is very fast, dull and stereotyped covering in 5 days, the dull and stereotyped growth rate of starting strain is 0.83mm/h, the dull and stereotyped growth rate of glossy ganoderma CCTCC M2013287 is 0.94mm/h, has improved 13.25%, sees accompanying drawing 4.Glossy ganoderma CCTCC M2013287 bacteria colony white, be just the fine hair shape, rear change flocculence, and felted in a measure, bacterium colony is thick.Reverse side slightly is micro-yellow, and slight milk fragrance is arranged.The form of liquid one, two generations seed is shown in accompanying drawing 5.
2, stability analysis
Take mycelial growth rate as index investigation stability, finishing screen is selected to preferably dissociant of 10 strains, through 5 times, go down to posterity, select optimum glossy ganoderma CCTCC M2013287 bacterial strain, this strain dissociant was gone down to posterity for 3 generations again, observe the variation of mycelial growth rate, and contrast with starting strain, thereby determine stability, then 3 generations of going down to posterity the results are shown in Table 3.
Table 3 is selected bacterial strain respectively for growth rate
As can be seen from Table 3, the growth rate of mutagenic strain JSU-10 (being glossy ganoderma JSU6161314) is higher than starting strain, its growth rate is degenerated not clearly, go down to posterity through 8 generations, still have the growth rate of 0.911mm/h, the character variation that glossy ganoderma produces after ultraviolet, microwave irradiation as can be seen here can heredity.
3. inheritance analysis
Antagonism is analyzed
Dissociant and starting strain are seeded on the PDA flat board simultaneously, and two bacterial strains are at a distance of certain distance, are placed in the incubator of 28 ℃ and cultivate, and observe the antagonism between bacterium colony after 4d.
Hypha of edible fungus not of the same race is in growing, and mycelia mutually restriction the other side's growth spreads, and produces antagonism, at intersection, forms the antagonism line, Here it is antagonism.Antagonism is not only in the existence not of the same race of edible mushroom, and of the same race but also exist between the discrepant bacterial strain of hereditary capacity, between bacterial strain, the power of antagonism has reflected the size of genetic diversity between bacterial strain, therefore antagonism not only can be for judging the affiliation between bacterial strain, and can, for breeding, identify whether bacterial strain produces variation.This test is connected to dull and stereotyped upper bacterium by starting strain and dissociant and has produced certain antagonism, certain variation has occurred in the growthform that can find out the right dissociant glossy ganoderma CCTCC M2013287 from Figure of description 6, its mycelial growth densification, and formed the antagonism line with left side starting strain, illustrated that starting strain and dissociant have produced genetic diversity.
Test two mutagenic strain glossy ganoderma CCTCC M2013287 and starting strain glossy ganoderma CFCC6043 output relatively
Mutagenic strain and starting strain through screening carry out ferment tank, relatively fermenting property.
Ganoderma strain capable adopts respectively mutagenic strain glossy ganoderma CCTCC M2013287 and starting strain glossy ganoderma CFCC6043.The fermentation tank sample-loading amount is 80% of fermentation tank volume, cultivation temperature is 28 ℃, ventilation 1:0.8v/v/mim, mixing speed 60r/min, tank gauge pressure 0.05MPa, inoculum concentration 8%, incubation time 5d, fermentation medium is rice bran 50g/L, wheat bran 30g/L, potassium dihydrogen phosphate 1.5g/L, magnesium sulfate 0.75g/L, pH nature.Weight method is measured the mycelia dry weight, and the phenol sulfuric acid process is measured mycelia polysaccharide.By the mycelia mixture of liquid culture gained, after centrifugation, then with distilled water washing 3 times, the culture fluid sticked to remove mycelium surface, put into air dry oven, under 60 ℃ of conditions, is dried to constant weight, obtains mycelia through weighing and mix dry weight.Add certain volume distilled water in the mycelia of drying, after grinding, lixiviate 3h in 100 ℃ of water-baths, quantitatively to a constant volume, measure mycelia mixing polysaccharide content by the phenolsulfuric acid method.Result of the test is: (1) mutagenic strain glossy ganoderma CCTCC M2013287 and starting strain glossy ganoderma CFCC6043 ferment in the same terms and medium, the mycelia dry weight is respectively 63.0g/L and 51.2g/L, and the mycelia dry weight of mutagenic strain glossy ganoderma CCTCC M2013287 has improved 23% than starting strain.(2) glossy ganoderma CCTCC M2013287 and a bacterial strain ferment in the same terms and medium, and both are respectively 2.03g/L and 1.40g/L mycelia polysaccharide, and the mycelia polysaccharide of glossy ganoderma CCTCC M2013287 has improved 45% than starting strain.Fermentation test shows, glossy ganoderma CCTCC M2013287 compares with starting strain glossy ganoderma CFCC6043, fermenting property and produce the mycelia polysaccharide performance good variation has occurred.
Claims (1)
1. for the bacterial strain of rice bran and wheat bran complete feed liquid state fermentation production GL-B, it is characterized in that being preserved in Chinese Typical Representative culture collection center, the Wuhan glossy ganoderma CCTCC M2013287 (Ganoderma lucidum CCTCC M2013287) of (being called for short CCTCC).
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